Abstract

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing are characterized. The studied variants can serve as the basis for the self-removal of polypeptide tags that can carry an affine label and optimize the process of the obtainment of target proteins and peptides in E. coli cells. They make it possible to synthesize target molecules composed of soluble and insoluble hybrid proteins (fusions), to conduct their affine purification and autocatalytic processing, and to obtain mature target products. The presented variants have a number of features as compared to the known prototypes. In particular, the mutant mini-intein Int4bPRO, which contains the L93P mutation, has temperature-dependent properties. It is able to produce target molecules composed of soluble fusions at a cultivation temperature below 30°C; however, it directs most synthesized fusions into insoluble intracellular aggregates after an increase in temperature to 37°C. The transition of Int4bPRO to an insoluble form is accompanied by the complete inactivation of C-terminal processing. Further application of the standard protein denaturation–renaturation procedures enables efficient reactivation of Int4bPRO and the processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, which contain the point mutation T62N or a combination of D144N and L146T mutations, respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows optimization of the biosynthesis of biologically active target proteins and peptides composed of soluble fusions that are suitable for affine purification and subsequent intein-dependent processing without the use of protein denaturation–renaturation procedures.

中文翻译：

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改良C-末端加工的产黄青霉微内含蛋白PRP8突变体的性质及生物技术应用

摘要

表征了来自产黄青霉（Int4b）的微型整合素PRP8的变异体，该变异体具有对C末端加工的改进控制。所研究的变体可作为自我标签的基础，该标签可带有仿射标签并优化在大肠杆菌中获得目标蛋白和多肽的过程。细胞。它们使合成由可溶性和不溶性杂合蛋白（融合物）组成的目标分子，进行仿射纯化和自催化过程以及获得成熟的目标产物成为可能。与已知的原型相比，提出的变体具有许多特征。尤其是，包含L93P突变的突变型迷你蛋白Int4bPRO具有温度依赖性。能够在低于30°C的培养温度下产生由可溶性融合物组成的目标分子；然而，在温度升高到37°C之后，它会将大多数合成的融合物引导到不溶的细胞内聚集物中。Int4bPRO转变为不溶形式的过程伴随着C末端加工的完全失活。标准蛋白质变性-变性程序的进一步应用可实现Int4bPRO的有效活化和体外融合处理。其他两个变体Int4b56和Int4b36分别包含点突变T62N或D144N和L146T突变的组合，其C末端加工的速率降低。它们用于大肠杆菌细胞可以优化生物活性靶蛋白和由可溶性融合物组成的肽的生物合成，这些融合物适用于仿射纯化和随后的依赖于intein的加工，而无需使用蛋白质变性-复性程序。